Neisseria gonorrhoeae is the causative agent of the sexually-transmitted disease gonorrhea as well as pelvic inflammatory disease, and disseminated gonococcal infection. Gonococci survive in the human population by generating a high degree of genetic diversity resulting in antigenic variation and the avoidance of the host immune response. The genetic diversity results from both intragenomic and intergenomic recombination. Intergenomic recombination results from natural transformation with DNA from other gonococci. Natural transformation is so frequent that the gonococcal population structure is considered panmictic. We discovered and are working to characterize a type IV secretion system (T4SS) in N. gonorrhoeae. We have shown that the T4SS acts to secrete chromosomal DNA, and that the secreted DNA is active in the transformation of other gonococci in the population. The T4SS is encoded on a genetic island present in eighty per cent of N. gonorrhoeae strains. Mutations in the T4SS genes resulted in loss of DNA secretion into the medium, drastic reduction in the ability of a strain to donate DNA for natural transformation, and altered interactions with host cells. In infection of primary cervical cells, T4SS mutants were delayed in attachment. At later times post-infection, the T4SS mutants were bound to the host cells as unusually large aggregates. Furthermore, studies with a transformed cervical cell line showed decreased invasion by a mutant deleted for the genetic island that encodes the T4SS. This proposal is focused on determining the function of the gonococcal T4SS - examining the mechanism of DNA secretion and how the secretion system affects host cell interactions. The goals of this proposal are contained in three specific aims. We will generate antibodies to specific proteins that make up the secretion apparatus and use these antibodies to determine the locations of the proteins in gonococcal cell compartments and on whole gonococcal cells. We will examine the mechanism of DNA secretion using genetic methods to identify and characterize proteins that act to bind and cleave DNA for secretion. We will characterize the effects of the T4SS on host cell infection using microscopy, invasion and intracellular growth assays, and measures of host cell signaling. We hypothesize that a secreted DNA-protein complex affects infection directly, by altering bacterial adherence or dispersion, or acts indirectly by affecting host cell signaling leading to different expression of host proteins affecting bacterial adherence, invasion, or intracellular survival. It is anticipated that these studies will not only lead to a better understanding of type IV secretion and the horizontal transfer of genes via transformation, but that they will also identify potential surface molecules that may be targets for drugs or immunotherapy. NARRATIVE: This project will characterize the mechanism of secretion of DNA and proteins by a secretion apparatus found in the bacterium Neisseria gonorrhoeae. These studies will lead to a better understanding of how genes are transferred between bacteria and how N. gonorrhoeae infects human cells.